The D-vitamins or calciferols arise from their provitamins through a cleavage, catalysed by sunlight, of the B-ring in the sterane rings. Their most important representatives are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol), which differ slightly only in the side chains, but which—so far as known—are similarly metabolised and have identical biological effects. Whereas provitamin D2 must be taken in with the food, the provitamin D3 can be formed in the human organism. So far as not more specifically designated by means of indices, the term vitamin D comprehends in the following in general all vitamin D forms. Vitamin D formed in the skin or taken in with food is bound in the plasma by vitamin D binding or transport proteins (DBP), transported to the liver and there metabolised to 25-hydroxy vitamin D (25-OH-D). The vitamin D binding protein DBP is also known as Gc-globulin or group specific component (J. G. Haddad in J. Steriod Biochem. Molec. Biol. (1995) 53, 579-582). Over 95% of the 25-hydroxy vitamin D measurable in the serum is as a rule 25-hydroxy vitamin D3. 25-Hydroxy vitamin D2 is only found in greater proportions if the person is receiving medication with vitamin D2 or, as is frequently the practice in the United States, foodstuffs are supplemented with vitamin D2.
25-Hydroxy vitamin D is the prevailing vitamin D metabolite in the blood circulation and its concentration in the serum generally indicates the vitamin D status, i.e. the extent to which vitamin D is available to the organism. If needed, 25-hydroxy vitamin D is metabolised in the kidneys to 1α,25-dihydroxy vitamin D, a hormone-like substance with great biological activity. The determination of 1α,25-dihydroxy vitamin D indicates how much vitamin D is present in the activated form.
The determination of 25-hydroxy vitamin D in a sample is preferably effected in accordance with the principle of competitive protein binding analysis, whereby on the basis of the displacement of radioactive 25-hydroxy vitamin D from the binding sites of a vitamin D binding protein, the 25-hydroxy vitamin D present in the sample can be quantified. Also, over the last several years, radioimmunoassays using 125I-labelled vitamin D derivatives and antibodies for vitamin D derivatives have established themselves in diagnosis. The data of the normal level of 25-hydroxy vitamin D in serum vary depending on the laboratory. It is, however, agreed that the concentration of 25-hydroxy vitamin D in the serum is as a rule greater than 5 ng/ml and smaller than 80 ng/ml. The competitive protein binding analysis requires the use of a radioactive vitamin D derivative which must have the same protein binding characteristics as 25-hydroxy vitamin D. The same applies also for the competitive binding analysis for the biologically active 1α,25-dihydroxy vitamin D and other vitamin D metabolites.
European patent specifications 0 312 360 and 0 363 211, and Tanabe et al. in J. Chem. Soc., Chem. Commun. 1989, 1220-1221 and J. Nutri. Sci. Vitaminol., 1991, 37, 139-147, disclose various 125I-labelled hydroxy- and dihydroxy vitamin D derivatives and their use in binding studies. These derivatives suffer the disadvantages that they are problematic to produce and are extremely labile. Light, radioactive rays, protons, hydrogen, enzymes, free radicals or the presence of iodine in free or bound form have great effect on the configuration and the binding characteristics of the vitamin D derivatives to vitamin D binding protein DBP or specific antibodies. Above all, they can cause or catalyse a rotation of the A-ring in the sterane system. The 3β-hydroxy-group of the vitamin D molecule is thereby rotated into the pseudo-1α-position, so that 5,6-trans-vitamin D is obtained. The so-called pseudo-1α-hydroxy-analogs of vitamin D may be metabolised similarly to vitamin D, but they have a structure which is different in significant points and are not bound or are significantly more poorly bound by vitamin D binding proteins such as for example DBP/Gc-Globulin or anti-vitamin D antibodies.

The above-described re-arrangement is to be understood as an example. Other chemical reactions and re-arrangements also occur. The same applies for 3H- or 14C-labelled vitamin D derivatives. These vitamin D derivatives are likewise not so stable that they permit a reliable binding analysis. The radioactive marking additionally increases the costs of storage, transport and disposal and is generally disadvantageous for health and the environment. Further the half-life of 125iodine is relatively short. On the other hand, a competitive binding analysis with 3H- and 14C-labelled vitamin D derivatives requires particular scintillation counters and is more demanding in terms of equipment, with largely the same problems.
Ray et al., in Biochemistry, 1991, 30, 4809-4813 disclose the coupling of vitamin D3 with various colouring groups. The detection sensitivity for dye-labelled vitamin D3 derivatives is, however, too small that one might use them in a competitive binding analysis for natural vitamin D metabolites, apart from the fact that the dye-labelled derivatives are not stable in serum and further are particularly light-sensitive.
Holick et al, describe in U.S. Pat. No. 5,981,779, issued on Nov. 9, 1999; and WO 97/24127, published on Jul. 10, 1997, with reference to Roy et al in Steroids (1995), 60(8), 530-533 a synthesis scheme said to produce 25-hydroxy-vitamin-D3-aminopropyl-3-(6-amino)-hexanoic acid and its conjugation with biotin-4-nitrophenylester. Example 6 describes that, on a molar basis, about 11 molecules of so synthesised biotin-vitamin-D-conjugate can displace one molecule of 3H-25-hydroxy-vitamin-D3 bound to human vitamin D binding protein DBP so that so synthesised molecules can be employed for an enzyme-linked immunosorption assay (ELISA) for measuring the concentration of vitamin D3 and 25-hydroxy-vitamin-D3 in a test solution.
It is the object of the invention to provide vitamin D derivatives, which are more efficient in displacing vitamin D metabolites such as 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D bound to vitamin D binding protein DBP to allow a more precise measurement of those by a competitive immunoassays.
This presumes the following properties: first, that for the vitamin D derivatives, a detection sensitivity exists which is higher than, or lies in a lower range of concentrations than, the concentration of the sought after vitamin D metabolites in the samples; second, that the derivatives are stable in serum, plasma or urine under the usual protonic conditions and are stable with the respect to serum enzymes; third, that the derivatives are sufficiently stable with regard to light and storage, over weeks and months, and finally, that the 25-hydroxy-group of the vitamin D derivative is intact and not tampered by the synthesis.